Endothelial cell-derived stem cell factor promotes lipid accumulation through c-Kit-mediated increase of lipogenic enzymes in brown adipocytes

Active thermogenesis in the brown adipose tissue (BAT) facilitating the utilization of lipids and glucose is critical for maintaining body temperature and reducing metabolic diseases, whereas inactive BAT accumulates lipids in brown adipocytes (BAs), leading to BAT whitening. Although cellular crosstalk between endothelial cells (ECs) and adipocytes is essential for the transport and utilization of fatty acid in BAs, the angiocrine roles of ECs mediating this crosstalk remain poorly understood. Using single-nucleus RNA sequencing and knock-out male mice, we demonstrate that stem cell factor (SCF) derived from ECs upregulates gene expressions and protein levels of the enzymes for de novo lipogenesis, and promotes lipid accumulation by activating c-Kit in BAs. In the early phase of lipid accumulation induced by denervation or thermoneutrality, transiently expressed c-Kit on BAs increases the protein levels of the lipogenic enzymes via PI3K and AKT signaling. EC-specific SCF deletion and BA-specific c-Kit deletion attenuate the induction of the lipogenic enzymes and suppress the enlargement of lipid droplets in BAs after denervation or thermoneutrality in male mice. These data provide insight into SCF/c-Kit signaling as a regulator that promotes lipid accumulation through the increase of lipogenic enzymes in BAT when thermogenesis is inhibited.

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Experiments were replicated at least once for all analyses to produce convincing results and the number of reproductions of each experimental finding is described in each figure legends. All attempts at experimental replication were successful.
Because the experiments required a specific genetic signature, randomization per treatment was not possible. The animal studies were performed using littermate controls. For the experiments of snRNA-seq, cold exposure, CL316,247 treatment, UDN, or thermoneutrality exposure, C57BL/6J mice were randomly chosen. Note that full information on the approval of the study protocol must also be provided in the manuscript. All the antibodies were validated for the species (mouse) and applications (immunohistochemistry and immunoblotting) by the correspondent manufacturer, which is described in the manufacturer's website. Our usage was described in the Methods section of the manuscript as below.
For immunofluorescence staining (IFS), the tissues were permeabilized and blocked with blocking buffer containing 5% donkey serum in 1% Triton-X 100 in PBS for 1 h at room temperature (RT). Then, they were incubated with a primary antibody diluted in the blocking buffer overnight at 4°C. After several washes with PBS, they were incubated with secondary antibodies (Jackson ImmunoResearch) diluted in the blocking buffer for 4 h at RT. After several washes with PBS, they were mounted with Vecta-shield (Vector Laboratories). For immunoblotting, membranes were incubated with 2% bovine serum albumin (BSA) blocking buffer for 1 h at RT, and blotted with the following antibodies overnight at 4°C. After several washes with TBST, they were incubated with secondary antibodies (Cell Signaling Technology) for 1 h at RT. Target proteins were detected using ECL western blot detection solution (#WBKLS0500, Millipore). The same amount of protein loading in each lane is verified by immunoblotting of tubulin or GAPDH. Mice were housed under a 12h light/dark cycle within a temperature-controlled room (21-22°C) and allowed to free access food (Teklad global 18% protein rodent diet, #2018C, Envigo®) and water The study dose not involve wild animals.
To avoid the off-target effect of tamoxifen on iWAT browning in the CreERT2 female mice (Zhao L et al., Int. J. Obes, 44:226-234, 2020) and to preclude the possible sexual differences among the mice groups, only male mice were used in all of this study.
The study does not involve samples collected from the field.
Animal care and experimental procedures were performed under the approval (KA2018-70) of the Institutional Animal Care and Use Committee of Korea Advanced Institute of Science and Technology (KAIST).